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  • 251.
    Eriksson, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Langström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Josephsson, Ray
    Assessment of receptor occupancy-over-time of two dopamine transporter inhibitors by [C-11]CIT and target controlled infusion2011In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 116, no 2, p. 100-106Article in journal (Refereed)
    Abstract [en]

    Introduction. Occupancy-over-time was determined for two dopamine transporter (DAT) inhibitors through modeling of their ability to displace the PET ligand [C-11]CIT. The tracer was held at a pseudo steady state in a reference tissue by target controlled infusion. Methods. Rhesus monkeys (n = 5) were given [C-11]CIT and studied with a PET scanner. Tracer uptake in the reference tissue cerebellum was held at a pseudo steady state by use of target controlled infusion. The pharmacokinetics/pharmacodynamics (PK/PD) of [C-11]CIT was assessed through the simplified reference tissue model (SRTM). Bupropion (n = 2) and GBR-12909 (n = 2) receptor occupancies were estimated through modeling of their effects on [C-11]CIT displacement. Results. There was a high uptake of [C-11]CIT in striatum, which contains a high DAT density. The reference tissue cerebellum had a comparatively low uptake. The modeling of [C-11]CIT PK/PD properties in striatum showed high binding potential (BP = 5.34 +/- 0.78). Both DAT inhibitors caused immediate displacement of [C-11]CIT after administration. The occupancy-over-time was modeled as a mono-exponential function, describing initial maximal occupancy (Occ(0)) and rate of ligand-receptor dissociation (k(off)). GBR-12909 showed irreversible binding (k(off) = 0) after an initial occupancy of 76.1%. Bupropion had a higher initial occupancy (84.5%) followed by a release half-life of 33 minutes (k(off) = 0.021). Conclusions. The proposed model can be used for assessment of in-vivo occupancy-over-time of DAT ligands by use of target controlled infusion of [C-11]CIT. The concept of assessing drug-receptor interactions by studying perturbations of a PET tracer from a pseudo steady state can be transferred to other CNS systems.

  • 252.
    Eriksson, Olof
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Radiology.
    Wallberg, Andreas
    Syvänen, Stina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Josephsson, Raymond
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Virology.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Bergström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    A computerized Infusion Pump for control of tissue tracer concentration during Positron Emission Tomography in vivo Pharmacokinetic/Pharmacodynamic measurements2008In: BMC Medical Physics, ISSN 1756-6649, E-ISSN 1756-6649, Vol. 8, no 2Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:

    A computer controlled infusion pump (UIPump) for regulation of target tissue concentration of radioactive compounds was developed for use in biological research and tracer development for PET.

    METHODS:

    Based on observed tissue or plasma kinetics after a bolus injection of the tracer an algorithm calculates the infusion needed to obtain a specified target kinetic curve. A computer feeds this infusion scheme into an infusion pump connected to an animal via a venous catheter. The concept was validated using [11C]Flumazenil administrated to Sprague-Dawley rats where the whole brain distribution and kinetic of the tracer was measured over time using a microPET-scanner. The accuracy and precision of the system was assessed by producing steady-state levels of the tracer and by mimicking kinetics after oral administration.

    RESULTS:

    Various kinetic profiles could be generated, including rapid achievement of constant levels, or step-wise increased levels. The resulting tissue curves had low deviation from the target curves according to the specified criteria: AUC (%): 4.2 +/- 2.8, Maximal deviation (%): 13.6 +/- 5.0 and R2: 0.95 +/- 0.02.

    CONCLUSION:

    The UIPump-system is suitable for use in PET-research for assessment of PK/PD properties by simulation of different tracer tissue kinetics in vivo.

  • 253. Eriksson, Per
    et al.
    Engman, Lars
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Organisk kemi.
    Lind, Johan
    Merényi, Gabor
    Aqueous Phase One-Electron Reduction of Sulfonium, Selenonium and Telluronium Salts2005In: Eur. J. Org. Chem., p. 701-705Article in journal (Refereed)
    Abstract [en]

    Triorganylsulfonium, -selenonium and -telluronium salts were reduced by carbon dioxide radical anions/solvated electrons produced in aqueous solution by radiolysis. The radical expulsion accompanying reduction occurred with the expected leaving group propensities (benzyl > secondary alkyl> primary alkyl> methy> phenyl), although greater than expected loss of the phenyl group was often observed. Diorganyl chalcogenides formed in the reductions were conveniently isolated by extraction with an organic solvent. Product yields based on the amount of reducing radicals obtained from the y-source were often higher than stoichiometric (up to 1800%) in the reduction of selenonium an dtelluronium compounds; it is likely that this result can be accounted for in terms of a chain reaction with carbon-centred radicals/formate serving as the chain transfer agent. The product distribution was essentially independent of the reducing species for diphenyl alkyl telluronium salts, whereas significant variations were seen for some of the corresponding selenonium salts. This would suggest the intermediacy of telluranyl radicals in the one-electron reduction of telluronium salts. However, pulse radiolysis experiments indicated that the lifetimes of such a species (the triphenyltelluranyl radical) would have to be less than 1 us.

  • 254.
    Eriksson, Sandra
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gutierrez Arenas, Omar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Bjerling, Pernilla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tomkinson, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Development, evaluation and application of tripeptidyl-peptidase II sequence signatures2009In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 484, no 1, p. 39-45Article in journal (Refereed)
    Abstract [en]

    Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

  • 255.
    Erlandsson, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Imaging of Enzymes in the Steroid Biosynthetic Pathway: Synthesis of 18F-Labelled Tracers2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis deals with the synthesis and development of 18F-labelled alkyl etomidate and vorozole analogues, and their use as positron emission tomography (PET) tracers for the imaging of the steroid enzymes 11β-hydroxylase and aromatase. Two synthetic 18F-labelling approaches to the etomidate and vorozole analogues were developed, and the analogues were evaluated in some biological assays.

    The two-step labelling method was used to synthesise many compounds for biological evaluation. In the first step, a 18F-labelled intermediate based on a ditosylate or a halogenated diethyl ether was synthesised and used directly in the next alkylation step. The decay-corrected (d.c.) radiochemical yield was higher compared to other known two-step labelling methods.

    Once an appropriate candidate has been chosen for clinical evaluation, a one-step labelling method will be more suitable. We therefore developed a method based on precursors that had leaving groups at the end of their alkyl chains, and used these directly in the 18F-labelling synthesis. The one-step 18F-labelling synthesis required less reaction time and produced higher specific radioactivity and d.c. radiochemical yield than our two-step synthesis. With microwave heating, the reaction time was reduced to seconds and the d.c. radiochemical yield was better than that obtained with conventional heating. The one-step synthesis simplified the technical handling by allowing the tracer syntheses to be automated on the TRACERLab FXFN.

    List of papers
    1. Synthesis of 11C-labelled metomidate analogues as adrenocortical imaging agents
    Open this publication in new window or tab >>Synthesis of 11C-labelled metomidate analogues as adrenocortical imaging agents
    2008 (English)In: J. Label Compd Radiopharm, no 51, p. 273-276Article in journal (Refereed) Published
    National Category
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-16689 (URN)doi:10.1002/jlcr.1517 (DOI)
    Available from: 2008-06-24 Created: 2008-06-24 Last updated: 2011-01-11
    2. 18F-Labelled Metomidate Analogues as Adrenocortical Imaging Agents
    Open this publication in new window or tab >>18F-Labelled Metomidate Analogues as Adrenocortical Imaging Agents
    2009 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 36, no 4, p. 435-445Article in journal (Refereed) Published
    Abstract [en]

    Introduction: Two- and one-step syntheses of 18F-labelled analogues of Metomidate, such as 2-[18F]fluoroethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (1), 2-[18F]fluoroethyl 1-[(1R)-1-(4-chlorophenyl)ethyl]-1H-imidazole-5-carboxylate (2), 2-[18F]fluoroethyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (3), 3-[18F]fluoropropyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (4) and 3-[18F]fluoropropyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (5) are presented.

    Methods: Analogues 1-5 were prepared by a two-step reaction sequence that started with the synthesis of either 2-[18F]fluoroethyl 4-methylbenzenesulfonate or 3-[18F]fluoropropyl 4-methylbenzenesulfonate. These were used as 18F-alkylating agents in the second step, in which they reacted with the ammonium salt of a 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylic acid. One-step-labelling syntheses of 1, 2 and 5 were also explored. Analogues 1-4 were biological validated by frozen-section autoradiography and organ distribution. Metabolite analysis was performed for 2 and 3.

    Results: The radiochemical yield of the two-step synthesis was in the range of 10-29%, and thatof the one-step synthesis was 25-37%. Using microwave irradiation in the one-step synthesis of 1 and 2 increased the radiochemical yield to 46 ± 3 and 79 ± 30%, respectively.

    Conclusion: Both the frozen-section autoradiography and organ distribution results indicated that analogue 2 has a potential as an adrenocortical imaging agent, having the highest degree of specific adrenal binding and best ratio of adrenal to organ uptake among the compounds studied.

    Keywords
    n. c. a. nucleophilic 18F-fluorination, [18F]alkyl MTO analogues, adrenocortical tumours
    National Category
    Chemical Sciences
    Research subject
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-89065 (URN)10.1016/j.nucmedbio.2009.01.014 (DOI)000266146700013 ()19423012 (PubMedID)
    Available from: 2009-02-06 Created: 2009-02-06 Last updated: 2017-12-14Bibliographically approved
    3. Synthesis and in vitro evaluation of 18F-labelled di- and tri(ethylene glycol) metomidate esters
    Open this publication in new window or tab >>Synthesis and in vitro evaluation of 18F-labelled di- and tri(ethylene glycol) metomidate esters
    2009 (English)In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 52, no 7-8, p. 278-285Article in journal (Refereed) Published
    Abstract [en]

    By replacing the alkyl chain in a metomidate ester with F-18-labelled   di- or tri(ethylene glycol) chains, two F-18-labelled PET tracers, i.e.  2-(2-[F-18]fluoroethoxy)ethyl 1-[(1R)-1-phenylethyll-1   H-imidazole-5-carboxylate (1) and   2-[2-(2-[F-18]fluoroethoxy)-ethoxylethyl   1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (2), were   synthesized. Two precursors, 2-(2-bromoethoxy)ethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate and   2-[2-(2-chloroethoxy)ethoxylethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate, were prepared and   used in one-step nucleophilic [F-18]fluorination reactions using   conventional and microwave heating. Organ distribution, frozen section   autoradiography and metabolite analysis were performed. The   decay-corrected radiochemical yields of 1 and 2 were 26 +/- 8 and 23   +/- 8%, respectively, when they were prepared using conventional   heating. By performing microwave heating, the reaction time could be   decreased and the yields of analogues 1 and 2 could be increased to 57   +/- 12 and 51 +/- 18%, respectively. Organ distribution studies in the   rat showed considerable uptake in the lungs, adrenals and liver. Both   compounds bound with low nonspecific binding (1: approx. 20-30%; 2:   2.9% or lower) to tissue from pig and human normal and pathologic   adrenals. Metabolite analyses were performed in rats after 5 and 30 min   for tracer 1 (20 +/- 6 and 2 +/- 1 %) and tracer 2 (27 +/- 5 and 6 +/-   4%). Both compounds are interesting candidates for the detection of   different types of adrenal disorders.

     

    Keywords
    n.c.a. nucleophilic 18F-fluorination, di- and tri(ethylene glycol), metomidate, analogues
    National Category
    Chemical Sciences
    Identifiers
    urn:nbn:se:uu:diva-88794 (URN)10.1002/jlcr.1597 (DOI)000268690300029 ()
    Available from: 2009-02-06 Created: 2009-02-06 Last updated: 2017-12-14Bibliographically approved
    4. 18F-Labelled vorozole analogues as PET tracer for aromatase
    Open this publication in new window or tab >>18F-Labelled vorozole analogues as PET tracer for aromatase
    2008 (English)In: J. Label Compd Radiopharm, no 51, p. 207-212Article in journal (Refereed) Published
    Keywords
    n. c. a. nucleophilic 18f-fluorination, 18F-labelled VOZ nalaogues, aromatase
    National Category
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-16607 (URN)doi:10.1002/jlcr.1502 (DOI)
    Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2011-01-11
    5. Pharmacological characterization of 18F-labeled vorozole analogues.
    Open this publication in new window or tab >>Pharmacological characterization of 18F-labeled vorozole analogues.
    Show others...
    (English)Manuscript (Other academic)
    Abstract [en]

    Two 18F-labeled analogues of vorozole ([18F]FVOZ and [18F]FVOO) have been developed as potential tools for the in vivo characterization of aromatase. The purpose of the project was to evaluate the pharmacological properties of these radioligands using a combination of in vitro binding and in vivo distribution studies in the rat and primate. Saturation binding studies with the radioligands in homogenates of rat ovary gave KD and Bmax values of 0.21 ± 0.1 nM and 210 ± 20 fmol/mg, respectively, for [18F]FVOZ, and 7.6 ± 1 nM and 293 ± 12 fmol/mg, respectively, for [18F]FVOO. Organ distribution studies in rats showed the highest accumulation in the adrenal glands, with standardized uptake values (SUVs) of 15 to 20, followed by ovaries and liver with SUVs of approximately 5. The SUVs in the remaining organs were between 0.5 and 1.5. There was probably some defluorination of both radioligands, as the accumulation of radioactivity in bone increased with time. The regional distribution in the brain was studied using ex vivo and in vitro autoradiography. In the brain, specific binding of both [18F]FVOZ and [18F]FVOO were found mainly in the amygdala. PET studies were performed in the Rhesus monkey, and these showed displaceable binding in the amygdala and the preoptic area of the hypothalamus. These studies suggest that [18F]FVOZ might be to be a suitable tracer for the study of aromatase in vitro and in vivo, and could be an alternative to [11C]vorozole in human PET-studies.

    Keywords
    aromatase, autoradiography, biodistribution, PET, rat brain, vorozole
    National Category
    Other Basic Medicine Medical and Health Sciences
    Research subject
    Organic Chemistry; Medicine
    Identifiers
    urn:nbn:se:uu:diva-88793 (URN)
    Available from: 2009-02-06 Created: 2009-02-06 Last updated: 2018-01-13
    6. Automated synthesis of 18F-labelled analogues of etomidate, vorozole and harmine using commercial synthesizer TRACERLab FXFN
    Open this publication in new window or tab >>Automated synthesis of 18F-labelled analogues of etomidate, vorozole and harmine using commercial synthesizer TRACERLab FXFN
    (English)Manuscript (Other academic)
    Abstract [en]

    18F-Labelled analogues of three biologically interesting compounds, ethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (ETO), 6-[(S)-(4-chlorophenyl)-(1H)-1,2,4-triazol-1-yl)methyl]-1-methyl-1H-benzotriazole (VOZ) and 7-methoxy-1-methyl-9H-β-carboline (HAR) were synthesized by one-step nucleophilic fluorination. The 18F-labelled products were obtained with 20–30% isolated decay-corrected radiochemical yields and the radiochemical purities were over 99% in all cases. The labelling syntheses were performed using fully automated commercial synthesizer TRACERLab FXFN. The automation of the syntheses of these three promising PET tracers using a commercial synthesizer will make them accessible for clinical applications.       

    Keywords
    PET, TRACERLab FXFN, ETO, VOZ, HAR, automation
    National Category
    Other Basic Medicine Organic Chemistry
    Research subject
    Organic Chemistry
    Identifiers
    urn:nbn:se:uu:diva-88789 (URN)
    Available from: 2009-02-06 Created: 2009-02-06 Last updated: 2018-01-13
  • 256.
    Erlandsson, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hall, Håkan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Synthesis and in vitro evaluation of 18F-labelled di- and tri(ethylene glycol) metomidate esters2009In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 52, no 7-8, p. 278-285Article in journal (Refereed)
    Abstract [en]

    By replacing the alkyl chain in a metomidate ester with F-18-labelled   di- or tri(ethylene glycol) chains, two F-18-labelled PET tracers, i.e.  2-(2-[F-18]fluoroethoxy)ethyl 1-[(1R)-1-phenylethyll-1   H-imidazole-5-carboxylate (1) and   2-[2-(2-[F-18]fluoroethoxy)-ethoxylethyl   1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (2), were   synthesized. Two precursors, 2-(2-bromoethoxy)ethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate and   2-[2-(2-chloroethoxy)ethoxylethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate, were prepared and   used in one-step nucleophilic [F-18]fluorination reactions using   conventional and microwave heating. Organ distribution, frozen section   autoradiography and metabolite analysis were performed. The   decay-corrected radiochemical yields of 1 and 2 were 26 +/- 8 and 23   +/- 8%, respectively, when they were prepared using conventional   heating. By performing microwave heating, the reaction time could be   decreased and the yields of analogues 1 and 2 could be increased to 57   +/- 12 and 51 +/- 18%, respectively. Organ distribution studies in the   rat showed considerable uptake in the lungs, adrenals and liver. Both   compounds bound with low nonspecific binding (1: approx. 20-30%; 2:   2.9% or lower) to tissue from pig and human normal and pathologic   adrenals. Metabolite analyses were performed in rats after 5 and 30 min   for tracer 1 (20 +/- 6 and 2 +/- 1 %) and tracer 2 (27 +/- 5 and 6 +/-   4%). Both compounds are interesting candidates for the detection of   different types of adrenal disorders.

     

  • 257.
    Erlandsson, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Karimi, Farhad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Lindhe, Örjan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    18F-Labelled Metomidate Analogues as Adrenocortical Imaging Agents2009In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 36, no 4, p. 435-445Article in journal (Refereed)
    Abstract [en]

    Introduction: Two- and one-step syntheses of 18F-labelled analogues of Metomidate, such as 2-[18F]fluoroethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (1), 2-[18F]fluoroethyl 1-[(1R)-1-(4-chlorophenyl)ethyl]-1H-imidazole-5-carboxylate (2), 2-[18F]fluoroethyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (3), 3-[18F]fluoropropyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (4) and 3-[18F]fluoropropyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (5) are presented.

    Methods: Analogues 1-5 were prepared by a two-step reaction sequence that started with the synthesis of either 2-[18F]fluoroethyl 4-methylbenzenesulfonate or 3-[18F]fluoropropyl 4-methylbenzenesulfonate. These were used as 18F-alkylating agents in the second step, in which they reacted with the ammonium salt of a 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylic acid. One-step-labelling syntheses of 1, 2 and 5 were also explored. Analogues 1-4 were biological validated by frozen-section autoradiography and organ distribution. Metabolite analysis was performed for 2 and 3.

    Results: The radiochemical yield of the two-step synthesis was in the range of 10-29%, and thatof the one-step synthesis was 25-37%. Using microwave irradiation in the one-step synthesis of 1 and 2 increased the radiochemical yield to 46 ± 3 and 79 ± 30%, respectively.

    Conclusion: Both the frozen-section autoradiography and organ distribution results indicated that analogue 2 has a potential as an adrenocortical imaging agent, having the highest degree of specific adrenal binding and best ratio of adrenal to organ uptake among the compounds studied.

  • 258.
    Erlandsson, Maria
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Karimi, Farhad
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Takahashi, Kayo
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Långström, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    18F-Labelled vorozole analogues as PET tracer for aromatase2008In: J. Label Compd Radiopharm, no 51, p. 207-212Article in journal (Refereed)
  • 259.
    Erlandsson, Maria
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Karimi, Farhad
    Yakahashi, Kayo
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    F-18-labelled vorozole analogues as PET tracer for aromatase.2008In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 51, no 5, p. 207-212Article in journal (Refereed)
    Abstract [en]

    One- and two-step syntheses for the F-18-labelling of 6-[(S)-(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-(2-[F-18]fluoroe thyl)-1H-benzotriazole, [F-18]FVOZ, 1 and 6-[(S)-(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-[2-(2-[18F]fluor oethoxy)ethyl]-1H-benzotriazole, [F-18]FVOO, 2 were developed. In the two-step synthesis, the nucleophilic fluorination step was performed by reacting (S)-6-[(4-chlorophenyl)-(1H-1,2,4-triazol-1-yl)methyl]-1H-benzotriazole (VOZ) with either the F-18-labelled ethane-1,2-diyl bis(4-methylbenzenesulfonate) or the oxydiethane-2,1-diyl bis(4-methylbenzenesulfonate). The radiochemical yields were in the range of 9-13% after the 110-120 min total syntheses and the specific radioactivities were 175 +/- 7 GBq/mu mol and 56 GBq/mu mol for compounds 1 and 2, respectively. In the one-step synthesis, the precursor 2-{6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-1,2,3-benzotriaz ol-1-yl}ethyl 4-methylbenzenesulfonate (7) or 1-[2-(2-bromoethoxy)ethyl]-6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)met hyl]-1H-benzotriazole (8) was directly labelled via, an 18F nucleophilic substitution to give the corresponding tracer. The labelled compounds were obtained in 36-99% radioichemical yield after 75-min syntheses. The specific radioactivities are 100 GBq/mu mol for compound 1 and 80 GBq/pmol for compound 2. In vitro autoradiography using frozen rat brains illustrated specific binding in the medial amygdala, the bed nucleus of stria terminalis and the preoptic area, all of which corresponded well to the result of C-11-labelled vorozole.

  • 260. Erondu, Ngozi
    et al.
    Gantz, Ira
    Musser, Bret
    Suryawanshi, Shailaja
    Mallick, Madhuja
    Addy, Carol
    Cote, Josee
    Bray, George
    Fujioka, Ken
    Bays, Harold
    Hollander, Priscilla
    Sanabria-Bohorquez, Sandra M.
    Eng, WaiSi
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hargreaves, Richard J.
    Burns, H. Donald
    Kanatani, Akio
    Fukami, Takehiro
    MacNeil, Douglas J.
    Gottesdiener, Keith M.
    Amatruda, John M.
    Kaufman, Keith D.
    Heymsfield, Steven B.
    Neuropeptide Y5 receptor antagonism does not induce clinically meaningful weight loss in overweight and obese adults2006In: Cell Metabolism, ISSN 1550-4131, Vol. 4, no 4, p. 275-282Article in journal (Refereed)
    Abstract [en]

    Neuropeptide Y (NPY) is a potent orexigenic neuropeptide, and antagonism of NPY Y1 and NPY Y5 receptors (NPYxR) is considered a potentially important anti-obesity drug target. We tested the hypothesis that blockade of the NPY5R will lead to weight loss in humans using MIK-0557, a potent, highly selective, orally active NPY5R antagonist. The initial series of experiments reported herein, including a multiple-dose positron-emission tomography study and a 12 week proof-of concept/dose-ranging study, suggested an optimal MK-0557 dose of 1 mg/day. The hypothesis was then tested in a 52 week, multicenter, randomized, double-blind, placebo-controlled trial involving 1661 overweight and obese patients. Although statistically significant at 52 weeks, the magnitude of induced weight loss was not clinically meaningful. These observations provide the first clinical insight into the human NPY-energy homeostatic pathway and suggest that solely targeting the NPY5R in future drug development programs is unlikely to produce therapeutic efficacy.

  • 261.
    Ersmark, Karolina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Nervall, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Gutiérrez-de-Terán, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hamelink, Elizabeth
    Janka, Linda K.
    Clemente, Jose C.
    Dunn, Ben M.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Samuelsson, Bertil
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Macrocyclic inhibitors of the malarial aspartic proteases plasmepsin I, II, and IV2006In: Biorganic & Medicinal Chemistry, no 14, p. 2197-2208Article in journal (Refereed)
  • 262.
    Ezzaher, Salah
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Bruhn, Clemens
    Ott, Sascha
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Directing protonation in [FeFe] hydrogenase active site models by modifications in their second coordination sphere2010In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 46, no 31, p. 5775-5777Article in journal (Refereed)
    Abstract [en]

    Subtle changes in the second coordination sphere of [Cl(2)bdtFe(2)-(CO)(4)(Ph2P-CH2-X-CH2-PPh2)] (bdt = benzene-1,2-dithiolate, X = NCH3, NCH2CF3, CH2) that do not influence the electronic character of the Fe-2 center can however direct protonation to three different sites: the N in the bis-phosphane, the Fe-Fe bond or the bdt-S.

  • 263. Fang, Yao-ren
    et al.
    MacMillar, Susanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Eriksson, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kolodziejska-Huben, Magdalena
    Dybala-Defratyka, Agnieszka
    Paneth, Piotr
    Matsson, Olle
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Westaway, Kenneth Charles
    The Effect of Solvent on the Structure of the Transition State for the SN2 Reaction between Cyanide Ion and Ethyl Chloride in DMSO and THF Probed with Six Different Kinetic Isotope Effects2006In: J. Org. Chem, no 71, p. 4742-4747Article in journal (Refereed)
    Abstract [en]

    The secondary a- and B-deuterium, the a-carbon, the nucleophile carbon, the nucleophile nitrogen, and the chlorine leaving group kinetic isotope effects forthe SN2 reaction between cyanide ion an dethyl chloride were determined in the very slightly polar solvent THF at 30 C. A comparison of these KIEs with those reported earlier for the same reaction in the polar solvent DMSO shows that the transition state in THF is only sligthly tighter with very slightly shorter NC-Ca-CI bonds. This minor change in transition state structure does not account for the different transition structures that were earlier suggested by interpreting the experimental KIEs and the gas-phase calculations, respectively. It therefore seems unlikely that the different transition states suggested by the two methods are due to the lack of appropriate solvent modeling in the theoretical calculations. Previously it was predicted that the transition state of SN2 reactions where the nucleophile and the leaving group have the same charge would be unaffected by a charge in solvent. The experimental KIEs support this view.

  • 264.
    Farkas, Viktor
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Rezeli, Melinda
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Vegvari, Akos
    Kilar, Ferenc
    Hjertén, Stellan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    General Approach for Certain Quantitative Calculations for Instance of the Variance of Reversible Adsorption to the Capillary Wall in CE2009In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 81, no 1, p. 343-348Article in journal (Refereed)
    Abstract [en]

    Miniaturization of analytical separation methods offers several advantages, including short run times, high resolution, and high recovery of the sample constituents. To optimize these parameters, the reversible adsorption (to minimize loss in resolution), as well as the irreversible adsorption (to minimize loss of analytes) must be quantified. However, no useful equation is available for the calculation of the variance of reversible adsorption. Therefore, we have taken another approach to quantify the reversible interaction. The method is unique and important since no equation for calculation of this variance is required. Instead, two experiments are required, which are run under such conditions that the variance of a certain parameter has the same numerical value in the two experiments (one with and without EOF), except for the variance of reversible adsorption. The approach is universal in the sense that it can be used for many Is different mathematical concepts and be modified to also cover certain functions other than a sum of parameters. We have also introduced a simple expression for the irreversible adsorption, which shows that the hydrophobic interaction from only two methyl groups in the coating gives rise to as much as 40-50% loss of protein, and the width of the zones in the capillary with this coating was 8-15% larger compared to the zone width in the polyacrylamide-coated capillaries. The reproducibility in migration time, peak area, and peak width in two consecutive runs in capillaries with two methyl groups in the coating was very low, but in EOF-free polyacrylamide-coated capillaries extremely high, indicating that the reversible and irreversible adsorption of proteins to this coating is negligible. The scanning detector, frequently used in free zone electrophoresis in the 1960s-1970s, gives true separation parameters and is, therefore, much preferable to the stationary detector used in most CE experiments, because this detector gives apparent separation parameters.

  • 265.
    Fedulova, Natalia
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Alpha-class Glutathione Transferases from Pig: a Comparative Study2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine.

    A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II).

    In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I).

    Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III).

     

    List of papers
    1. Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization
    Open this publication in new window or tab >>Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization
    2010 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 431, no 1, p. 159-167Article in journal (Refereed) Published
    Abstract [en]

    A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

    Keywords
    Delta(5)-androstene-3, 17-dione, androstenedione, double-bond isomerization, glutathione transferase Alpha 2-2 (GST A2-2), steroidogenesis, tributyltin
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-134177 (URN)10.1042/BJ20100839 (DOI)000282852800017 ()20673231 (PubMedID)
    Available from: 2010-11-22 Created: 2010-11-22 Last updated: 2017-12-12Bibliographically approved
    2. Characterization of porcine Alpha-class glutathione transferase A1-1
    Open this publication in new window or tab >>Characterization of porcine Alpha-class glutathione transferase A1-1
    2011 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 507, no 2, p. 205-211Article in journal (Refereed) Published
    Abstract [en]

    An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

    Keywords
    Sus scrofa, glutathione transferase A1-1, alternative sequence, substrate selectivity
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-140698 (URN)10.1016/j.abb.2010.12.015 (DOI)000288058000001 ()21172301 (PubMedID)
    Available from: 2011-01-07 Created: 2011-01-07 Last updated: 2017-12-11Bibliographically approved
    3. Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases
    Open this publication in new window or tab >>Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases
    2011 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed) Published
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

    Keywords
    concentration-dependent instability, functional comparison, glutathione transferase, reproducibility, time-dependent instability
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-144107 (URN)10.1016/j.ab.2011.01.041 (DOI)000289607900003 ()21295006 (PubMedID)
    Funder
    Swedish Research Council
    Available from: 2011-01-27 Created: 2011-01-27 Last updated: 2017-12-11Bibliographically approved
  • 266.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hanrieder, Jörg
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Bergquist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Analytical Chemistry.
    Emrén, Lars O.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Expression and purification of catalytically active human PHD3 in Escherichia coli2007In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 54, no 1, p. 1-10Article in journal (Refereed)
    Abstract [en]

    Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after heterologous expression in Escherichia coli. Histidine-tagged enzyme was isolated as monomer by immobilized Ni-affinity chromatography followed by gel filtration. Overexpression of bacterial chaperonins GroEL/ES at 30 °C substantially increased the yield of soluble PHD3. High concentrations of salt and reducing agent during purification prevented protein aggregation. The enzyme activity with peptide derived from HIF-1α was inhibited by Zn2+, desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1α was discovered as a new site of hydroxylation.

  • 267.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Experimental Conditions Affecting Functional Comparison of Highly Active Glutathione Transferases2011In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 413, no 1, p. 16-23Article in journal (Refereed)
    Abstract [en]

    Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

  • 268.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Raffalli-Mathieu, Francoise
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Characterization of porcine Alpha-class glutathione transferase A1-12011In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 507, no 2, p. 205-211Article in journal (Refereed)
    Abstract [en]

    An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

  • 269.
    Fedulova, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Raffalli-Mathieu, Francoise
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Porcine glutathione transferase Alpha 2-2 is a human GST A3-3 analogue that catalyses steroid double-bond isomerization2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 431, no 1, p. 159-167Article in journal (Refereed)
    Abstract [en]

    A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

  • 270. Feil, S. C.
    et al.
    Tang, Julian
    Hansen, Guido
    Gorman, A
    Wiktelius, Eric
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Stenberg, Gun
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Parker, Michael W.
    Crystallization and preliminary X-ray analysis of glutathione transferases from cyanobacteria2009In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 65, p. 475-477Article in journal (Refereed)
    Abstract [en]

    Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

  • 271.
    Filonova, Lada
    et al.
    Institutionen för skogens produkter, trävetenskap, Sveriges lantbruksuniversitet.
    Kallas, Åsa M.
    Greffe, Lionel
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Teeri, Tuula T.
    Daniel, Geoffrey
    Institutionen för skogens produkter, trävetenskap, Sveriges lantbruksuniversitet.
    Analysis of the Surfaces of Wood Tissues and Pulp Fibers Using Carbohydrate-Binding Modules Specific for Crystalline Cellulose and Mannan2007In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 8, no 1, p. 91-97Article in journal (Refereed)
    Abstract [en]

    Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1HjCel7A) and mannans (CBM27TmMan5 and CBM35CjMan5C) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1HjCel7A was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1HjCel7A revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27TmMan5 and CBM35CjMan5C CBMs were found to be more specific reagents than a monoclonal antibody specific for (1→4)-β-mannan/galacto-(1→4)-β-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.

  • 272. Fogarty, Heather A.
    et al.
    Ottosson, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Michl, Josef
    Calculation of relative energies of permethylated oligosilane conformers in vapor and in alkane solution2006In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 110, no 50, p. 25485-25495Article in journal (Refereed)
    Abstract [en]

    The geometries of 35 conformers of Me(SiMe2) nMe (n= 4, 1; n = 5, 2; n = 6, 3; n = 7, 4) were optimized at the MP2/VTDZ level, and CCSD(T) single-point calculations were done at three MP2/VTDZ conformer geometries of 1. The relative ground-state energies of the conformers of 1-4 in the gas phase were obtained from the MP2/VTDZ electronic energy, zero- point vibrational energy, and thermal corrections at 0, 77, and 298 K. Relative energies in an alkane solvent at 77 and 298 K were obtained by the addition of solvation energies, obtained from the SM5.42R model. The calculated energies of 26 of the conformers (n = 4-6) have been least-squares fitted to a set of 15 additive increments associated with each Si-Si bond conformation and each pair of adjacent bond conformations, with mean deviations of 0.06-0.20 kcal/mol. An even better fit for the energies of 24 conformers (mean deviations, 0.01-0.09 kcal/mol) has been obtained with a larger set of 19 increments, which also contained contributions from selected combinations of conformations of three adjacent bonds. The utility of the additive increments for the prediction of relative conformer energies in the gas phase and in solution has been tested on the remaining nine conformers (n = 6, 7). With the improved increment set, the average deviation from the SM5.42R//MP2 results for solvated conformers at 298 K was 0.18 kcal/mol, and the maximum error was 0.98 kcal/mol.

  • 273.
    Fondell, Amelie
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Ickenstein, Ludger M
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Physical Chemistry.
    Sjöberg, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Carlsson, Jörgen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Gedda, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences.
    Nuclisome: a novel concept for radionuclide therapy using targeting liposomes2010In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 37, no 1, p. 114-123Article in journal (Refereed)
    Abstract [en]

    PURPOSE: For the treatment of cancer, the therapeutic potential of short-range, low-energy Auger-electron emitters, such as (125)I, is getting progressively wider recognition. The potency of Auger-electron emitters is strongly dependent on their location in close vicinity to DNA. We have developed a new two-step targeting strategy to transport (125)I into cancer-cell nuclei using PEG-stabilized tumour-cell targeting liposomes named "Nuclisome-particles". METHODS: In the present study, epidermal growth factor (EGF) was used as a tumour-cell-specific agent to target the EGF-receptor (EGFR) and the liposomes were loaded with (125)I-Comp1, a recently synthesized daunorubicin derivative. RESULTS: As analysed with cryo-TEM, the derivative precipitates inside liposomes at a drug-to-lipid molar ratio of 0.05:1. Receptor-specific uptake in cultured U-343MGaCl2:6 tumour cells of EGFR-targeting liposomes increased with time while non-specific and receptor-blocked uptake remained low. Nuclisome-particles were able to target single U-343MGaCl2:6 cells circulating in human blood during 4 h, with low uptake in white blood cells, as demonstrated in an ex vivo system using a Chandler loop. Autoradiography of targeted cells indicates that the grains from the radiolabelled drug are mainly co-localized with the cell nuclei. The successful targeting of the nucleus is shown to provide high-potency cell killing of cultured U-343MGaCl2:6 cells. At the concentration used, Nuclisome-particles were up to five orders of magnitude more effective in cell killing than EGFR-targeting liposomes loaded with doxorubicin. CONCLUSION: The results thus provide encouraging evidence that our two-step targeting strategy for tumour cell DNA has the potential to become an effective therapy against metastasizing cancer cells in the bloodstream.

  • 274. Forngren, Tobias
    et al.
    Samuelsson, Linda
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Organisk kemi.
    Långström, Bengt
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry II. Organisk kemi.
    A 11C-methylstannane (5-[11C]methyl-1-aza-5-stannabicyclo[3.3.3] undecane) for use in palladium-mediated [11C]C-Cbond forming reactions with organic halides2004In: J. Labelled Compd. Rad., no 47, p. 71-78Article in journal (Refereed)
  • 275. Forsberg, A
    et al.
    Almkvist, O
    Engler, Henry
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Section of Nuclear Medicine and PET.
    Wall, Anders
    Uppsala Imanet AB, GE Healthcare, Uppsala, Sweden.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nordberg, A
    High PIB Retention in Alzheimer's Disease is an Early Event with Complex Relationship with CSF Biomarkers and Functional Parameters2010In: Current Alzheimer Research, ISSN 1567-2050, Vol. 7, no 1, p. 56-66Article in journal (Refereed)
    Abstract [en]

    Background:

    New in vivo amyloid PET imaging tracers, such as 11C-PIB, provide possibilities to deeper understand the underlying pathological processes in Alzheimers disease (AD). In this study we investigated how 11C-PIB retention is related to cerebral glucose metabolism, episodic memory and CSF biomarkers.

    Method:

    Thirty-seven patients with mild AD and 21 patients with mild cognitive impairment (MCI) underwent PET examinations with the amyloid tracer 11C-PIB, 18F-FDG for measurement of regional cerebral metabolic rate of glucose (rCMRglc), assessment of episodic memory and assay of cerebral spinal fluid (CSF) levels of amyloid-ß (Aβ1-42), total tau and phosphorylated tau respectively. Analyses were performed using Statistical Parametric Mapping (SPM) and regions of interest (ROIs).

    Results:

    Pooled data from AD and MCI patients showed strong correlations between 11C-PIB retention, levels of CSF biomarkers (especially Aß1-42), rCMRglc and episodic memory. Analysis of the MCI group alone revealed significant correlations between 11C-PIB retention and CSF biomarkers and between CSF biomarkers and episodic memory respectively. A strong correlation was observed in the AD group between rCMRglc and episodic memory as well as a significant correlation between 11C-PIB retention and rCMRglc in some cortical regions. Regional differences were observed as sign for changes in temporal patterns across brain regions.

    Conclusions:

    A complex pattern was observed between pathological and functional markers with respect to disease stage (MCI versus AD) and brain regions. Regional differences over time were evident during disease progression. 11C-PIB PET and CSF Aß1-42 allowed detection of prodromal stages of AD. Amyloid imaging is useful for early diagnosis and evaluation of new therapeutic interventions in AD.

  • 276. Forsberg, Anton
    et al.
    Engler, Henry
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Almkvist, Ove
    Blomquist, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Hagman, Goran
    Wall, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology.
    Ringheim, Anna
    Langström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Nordberg, Agneta
    PET imaging of amyloid deposition in patients with mild cognitive impairment2008In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 29, no 10, p. 1456-1465Article in journal (Refereed)
    Abstract [en]

    mild cognitive impairment, converters, amyloid, PET, PIB, FDG, CSF biomarkers/k It is of great clinical value to identify Subjects at a high risk of developing AD. We previously found that the amyloid positron emission tomography (PET) tracer PIB showed a robust difference in retention in the brain between AD patients and healthy controls (HC). Twenty-one patients diagnosed with MCI (mean age 63.3 +/- 7.8 (S.D.) years) Underwent PET Studies with C-11-PIB, and F-18-fluoro-deoxy-glucose (FDG) to measure cerebral glucose metabolism, its well as assessment of cognitive function and CSF sampling. Reference group data from 27 AD patients and 6 healthy controls, respectively. were Used for comparison. The mean cortical PIB retention for the MCI patients was intermediate compared to HC and AD. Seven MCI patients that later Lit clinical follow-up converted to AD (8.1 +/- 6.0 (S.D.) months) showed significant higher PI B retention compared to non-converting MCI patients and HC, respcctively (ps < , 0.01). The PIB retention in MCI converters was comparable to AD patients (p > , 0.01). Correlations were observed in the MCI patients between PI B retention and CSF A beta(1-42). total Tau and episodic memory, respectively.

  • 277. Frank, Richard A.
    et al.
    Långström, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Antoni, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
    Montalto, Michael C.
    Agdeppa, Eric D.
    Mendizabal, Marivi
    Wilson, Ian A.
    Vanderheyden, Jean-Luc
    The Imaging Continuum: Bench to Biomarkers to Diagnostics2007In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 50, no 9-10, p. 746-769Article, review/survey (Refereed)
    Abstract [en]

    Innovation in basic and applied science has brought radiotracers to fruition as diagnostics. Non-invasive, longitudinal, and quantifiable molecular imaging is the key to diagnosing and monitoring numerous illnesses, with more to come from characterization of the clinical relevance of findings from genomics research. Radiotracers enable real-time in vivo studies of the effects of drug candidates on receptors, pathways, pharmacodynamics, and clinically relevant endpoints, thereby providing both early detection of pathophysiology to enable early intervention, and then monitoring of treatment responses to enable individualization of treatment regimens. We review developments which have translated imaging from bench to bedside, or biomarkers to diagnostics. Notable developments include (1) synthesis methods for rapid 11C labeling of biomolecules to high specific radioactivity; (2) ligand-binding assays for screening molecular imaging agents rather than drugs; (3) in vivo imaging of radiotracers in animals; (4) discovering the imaging advantages of 99mTc, 11C, and 18F; (5) co-registration and automated quantitative assessment of high spatial resolution CT and MR images with molecular images from PET for longitudinal studies of treatment effect.

  • 278.
    Furmark, Tomas
    et al.
    Uppsala University, Humanistisk-samhällsvetenskapliga vetenskapsområdet, Faculty of Social Sciences, Department of Psychology.
    Tillfors, Maria
    Uppsala University, Humanistisk-samhällsvetenskapliga vetenskapsområdet, Faculty of Social Sciences, Department of Psychology.
    Garpenstrand, Håkan
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Neuroscience.
    Marteinsdottir, Ina
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Neuroscience.
    Långström, Bengt
    Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry and Organic Chemistry. Uppsala Imanet.
    Oreland, Lars
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Neuroscience.
    Fredrikson, Mats
    Uppsala University, Humanistisk-samhällsvetenskapliga vetenskapsområdet, Faculty of Social Sciences, Department of Psychology.
    Serotonin transporter polymorphism related to amygdala excitability and symptom severity in patients with social phobia2004In: Neuroscience Letters, Vol. 362, p. 189-192Article in journal (Refereed)
  • 279.
    Garg, Neeraj
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gogoll, Adolf
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry.
    Westerlund, C
    Sundell, S
    Karlen, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Preparation of cis-5-methoxy-and-7-methoxy-1-acetoxy-1,2,3,4,4a,10a-hexahydro-9(10H)-phenanthrenone. An epoxide-arene reaction involving a selective 1,2-alkyl shift rearrangement1996In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 52, no 48, p. 15209-15224Article in journal (Refereed)
    Abstract [en]

    The preparation of cis-1α-acetoxy-7-methoxy-1,2,3,4,4a,10a-hexahydro-9(10H)- phenanthrenone 5 was accomplished starting from 6-methoxy-1-tetralone. Reduction of 7-methoxy-1,2,3, 4,9,10-hexahydro-1-oxo-phenanthrene 8, acetylation and subsequent oxidation delivered 5. Application of an analogus procedure to the preparation of cis-1β-acetoxy-5-methoxy-1,2,3,4,,4a,10a-hexahydro-9(10H)- phenanthrenone 6 was not feasible. A more elaborate route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation.

    The compounds 5 and 6 were prepared. A route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation.

  • 280.
    Gayet, Arnaud
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Kinetic resolution of racemic epoxides using a chiral diamine catalyst2005In: Tetrahedron Letters, no 46, p. 4805-4807Article in journal (Refereed)
  • 281.
    Gayet, Arnaud
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Bertilsson, Sophie
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Andersson, Pher
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I.
    Novel Catalytic Kinetic Resolution of Racemic Epoxides to Allylic Alcohols2002In: Organic Letters, Vol. 4, no 22, p. 3777-3779Article in journal (Refereed)
  • 282.
    Gayet, Arnaud
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Organisk kemi.
    Bolea, Christine
    Andersson, Pher
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Chemistry. Department of Biochemistry and Organic Chemistry, Organic Chemistry I. Organisk kemi.
    Development of new camphor based N,S chiral ligands and their application in transfer hydrogenation2004In: Organic & Biomolecular Chemistry, no 2, p. 1887-1893Article in journal (Refereed)
  • 283.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Dahl, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Kinetic characterization of HCV NS3 inhibitors using an SPR biosensorManuscript (Other academic)
    Abstract [en]

    An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied.

    The KD of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between koff or KD with Ki (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.

  • 284.
    Geitmann, Matthis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Dahl, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Mechanistic and kinetic characterization of hepatitis C virus NS3 protein interactions with NS4A and protease inhibitors2011In: Journal of Molecular Recognition, ISSN 0952-3499, E-ISSN 1099-1352, Vol. 24, no 1, p. 60-70Article in journal (Refereed)
    Abstract [en]

    The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.

  • 285.